Identification of novel SNPs in SYK gene of breast cancerpatients: computational analysis of SNPs in the 50UTR

Sehrish Kanwal • Mahmood Akhtar Kayani •

Rani Faryal

Received: 15 September 2011 / Accepted: 5 June 2012 / Published online: 16 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Spleen tyrosine kinase (SYK) is a non receptor

type tyrosine kinase and a known candidate tumor sup-

pressor gene in breast carcinoma. Loss of Syk is associated

with breast cancer invasion and increased cell mortality.

The main goal of our study was to detect germ-line poly-

morphisms in SYK gene in breast cancer affected females

of Pakistani origin, in order to understand the genetic basis

of complex human breast cancer. Seven novel SYK gene

SNPs were identified in breast cancer patients. Among

these, three were identified in intronic region, one at the

50splice donor site (50SD) and three in 50untranslated region

(50UTR) of SYK gene. Mutations at the 50SD and at 50UTR

can be crucial and could be responsible for generation of

mutated Syk protein. In silico analysis of the 50UTR vari-

ations revealed that one of the mutations was responsible

for generation of a more stable structure of 50UTR. Such a

change in pre-mRNA could potentially down regulate SYK

expression. These findings add to the growing literature

implicating dysfunctional SYK gene as a contributor to

human breast cancer, and suggest that therapies targeting

its molecular pathways could provide effective means of

treating/preventing breast cancer.

Keywords Novel SNPs � 50UTR � SYK gene �Breast cancer

Introduction

Breast cancer is one of the leading and devastating types of

cancer in women all around the globe with a prevalence of

approximately 12.6 % women all over the world [1, 2].

Pakistan, on the other hand, exhibits its highest ratio among

all Asian populations with lifetime prevalence of 1 in every

9 women [3, 4]. It is characterized by alternations of the

genes that influence cellular pathways and are involved in

growth and development [5].

Protein tyrosine kinases have been identified for their

critical role in breast cancer development [6]. Spleen tyro-

sine kinase, one of the two members of the protein tyrosine

kinase family ZAP-70/Syk, is a well known candidate

tumor suppressor gene. Role of SYK has been well docu-

mented in down regulation of breast cancer [7–9]. Some

scientists believe that the tumor suppressive activity of Syk

is both by enhancement of cell–cell interactions and

decreasing cellular motility [10], while according to others

it is due to abnormal mitotic progression and cell death

[11–13]. Carcinoma cells transfected with SYK cDNA

display decreased tumorigenicity [7, 13, 14]. Several stud-

ies have reported the expression of Syk in normal breast

cells and in benign breast tumors, but not in invasive car-

cinoma [14, 15]. This suggests the use of SYK as a valuable

biomarker for early breast cancer lesion detection [9, 16].

Loss of SYK expression in invasive breast cancer has

remained unclear. Most of the studies show the hyperme-

thylation of CpG islands of SYK gene promoter region to

be its major cause [7]. In normal breast cells, full length

Syk (L) form is expressed, which after alteration due to

hypermethylation of its promoter region loses normal

expression, resulting in shorter survival [17]. Some studies

have shown that aberrant RNA splicing of SYK gene

generates shorter Syk (S) form lacking a 23 residue

S. Kanwal � M. A. Kayani � R. Faryal (&)

Molecular Medicine Laboratory, Department of Biosciences,

COMSATS Institute of Information Technology, Park Road,

Islamabad, Pakistan

e-mail: ranifaryal@comsats.edu.pk

123

Mol Biol Rep (2012) 39:8345–8351

DOI 10.1007/s11033-012-1684-y

sequence in Interdomain B [18]. Syk (S) has been observed

active only in breast cancer cells and not in normal

mammary cells. Despite having resemblance, both forms of

SYK gene function differently [18].

To date, a lot of work has been done on SYK methyl-

ation levels in breast cancer. In the present study, SYK

germ-line mutations were investigated in breast cancer

patients of Pakistani origin, using single-strand conforma-

tional polymorphism (SSCP) analysis. Additionally com-

putational analysis was performed for the identified SNPs

to predict structural changes in pre-mRNA, which can

effectively play a role in the pathogenesis of cancer by

altering SYK gene expression.

Materials and methods

Identification of patients and sample collection

This study consisted of 120 breast cancer cases along with

120 age- and gender-matched disease free individuals as

controls. Blood samples were collected after informed con-

sent from patients by visiting National Oncology Research

Institute (NORI), Islamabad, Pakistan. Patient’s history was

obtained for epidemiological analysis with current ethical

standards and in accordance with international rules and

regulation. Prior approval was obtained from the ethical

committee of Department of Biosciences, COMSATS

Institute of Information Technology, Islamabad, Pakistan.

Genomic DNA extraction

DNA was extracted using established phenol–chloroform

extraction method [19].

Polymerase chain reaction

Primer3 software was used to design primers based on

sequence information obtained from publicly available

database (Enesembl). DNA was amplified with designed

primers with optimized conditions of PCR reagents. For

mutational screening, PCR was carried out for 35 cycles

(Perkin Elmer thermocycler, Veriti system, Applied Bio-

systems, USA). Thermal cycling conditions were denatur-

ation at 95 �C for 5 min, 35 cycles of 94 �C for 45 s, 58 �C

for 45 s, 72 �C for 45 s, and then a final extension at 72 �C

for 7 min.

Single strand conformational polymorphism (SSCP)

and DNA sequencing

For SSCP, thermal shock was carried for PCR products

along with denaturing reagent. After that PCR products

were run on an 8 % PAGE gel at 120 volts for about 2–3 h.

The gel was visualized with Quantity One software

package (BioRad, UK) after ethidium bromide staining

(1.0 lg/mL) using a UV trans-illuminator DOC-XR gel

documentation system (BioRad, UK). Samples that showed

band variations were cross checked with control and then

selected samples were sequenced from MC LAB, USA.

Sequencing results were visualized by BioEdit Sequence

Alignment Editor Version 7.0.0 software.

Statistical analysis

For statistical analysis, Chi-square test was used using SPSS

version 16 (SPSS Inc., Chicago, IL, USA), where P values

less than 0.05 were considered statistically significant.

50UTR structure prediction

Using the bioinformatics tool, UNAFOLD [20],

50untranslated region (50UTR) structure was predicted with

Table 1 Socio-demographical features of controls and breast cancer

patients of the Pakistani population

Patients Control

No. 120 120

Mean age in years 45.7 42.3

Socio-economic status (%)

High 10 13

Middle 57.9 65

Low 32.1 22

Menstrual status (%)

Pre 24.2 46.3

Post 75.7 53.7

Mean age at menarche in years (SD) 11.9 ± 0.8 12.2 ± 0.8

Early menarche (\11 years) (%) 15.6 12.3

Nulliparity (%) 10.7 39.8

Means number of pregnancies (SD) 3.3 ± 2.3 3.1 ± 2.6

Age at first full term pregnancy (SD) 19 ± 1.1 22 ± 1.5

Age at menopause (%)

\51 years 53.1 55.4

[51 years 30 21.6

None 16.9 34

Family history of cancer (%) 7.6 4.1

Any cancer 53.1 61

Breast cancer 30 17

None 16.9 32

Breast involved (%)

Right 44.3 –

Left 50 –

Both 5.7 –

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123

user defined parameters (Tm = 50 �C and Na? = 15 mM,

respectively), to evaluate possible effects of sequence

variations on the structural stability of SYK pre-mRNA. To

predict a stable mRNA that remains intact at body tem-

perature, specific conditions were set according to average

concentration of Na? in human cell (15 mM) and normal

human body temperature 37 �C [21].

Results

Socio-demographical features

A total of 120 patients were recruited from the National

Oncology Research Institute (Islamabad, Pakistan) with

age ranging from 20 to 85 years. Average age at diagnosis

of disease was 45.7 years. Most patients belonged to

middle class, were married and very few were gainfully

employed. Nearly 7.6 % patients had a family history of

cancer with invasive bilateral breast cancer. Most patients

had reached early menopause before the age of 50. Addi-

tional socio-demographical features of these patients are

listed in Table 1.

SYK germ-line mutations identified in patients

with breast cancer

In the present study, novel single nucleotide alterations (7)

were observed in 120 breast cancer patients (Figs. 1, 2).

Patients with SYK germ-line mutations were slightly

Fig. 1 SSCP and DNA

sequencing results of exon 4 of

SYK gene. a SSCP analysis

showing band shift variation.

b PCR products after

sequencing show SNPs at six

different positions (Panels a–k)

show mutations, resulting from

G ? A transition (26,353

G[A; 26,367 G[A; 26,414

G[A; 26,423 G[A; 26,484

G[A; 26,531 G[A)

Mol Biol Rep (2012) 39:8345–8351 8347

123

younger and had early or no menopause. Among observed

mutations, 3 were identified in coding region of exon 4,

encoding untranslated region (50UTR) and 4 in intronic

sequences of SYK. Among the observed intronic single

nucleotide polymorphisms (SNPs), one was identified in

intron 4 at position 26,353 G[A and two in intron 5 at

position 26,484 G[A and 26,531 G[A and one at 63,540

T[G, with a frequency of 35.8, 49.1, 33.3 and 80.0 %

respectively (Table 2). Three coding region mutations were

identified at position 26,367 G[A, 26,414 G[A and 26,423

G[A with a frequency of 68.3, 30.0 and 56.6 %, respec-

tively. These SNPs have not been reported either in the

SNP database, NCBI or Ensemble for any disease.

In silico prediction of 50UTR

To analyze effects of the identified SNPs in SYK expres-

sion, structural stability of SYK transcripts was determined

by the prediction of its 50UTR structure (original versus

mutated sequences) (Fig. 3). By comparing values of DG

(possibility of forming a secondary structure), their melting

temperature (Tm) and length of stem loop along with

GC/AT content in stem loop of mutated and original

structure, it was observed that the mutated SYK RNA

structure was more stable as compared to the non-mutated

one (Table 3). When mutations were tested individually, it

was observed that mutation at 26,414 G[A was responsible

for the structural stability of SYK.

Discussion

Breast cancer is the most common malignancy of females

and second leading cause of mortality due to cancer among

women after lung cancer [22]. Key factors that affect breast

carcinoma development include role of environment and

genetics, effect of endogenous and exogenous hormones,

host vulnerability, reproductive experience and biological

determinants of breast cancer [4]. This study was con-

ducted for identification and in silico analysis of SYK

germ-line mutations in the Pakistani population.

In this study, it was observed that breast cancer is more

prevalent in the age group 40–49 years (36.6 % patients)

followed by 50–59 years (30 % cases). These finding are in

concordance with other studies conducted in various region

of Pakistan [4, 23]. Left breast involvement was observed

in 59 % patients, right breast in 45 %, while 5.8 % were

with bilateral breast cancer. These findings were in line

with early findings at San Francisco, USA and in Punjab,

Pakistan [24, 25].

Contrary to the West, in Pakistan the incidence of breast

cancer increases with age, but after menopause its rate

decreases [25]. In our study, a maximum number of patients

were diagnosed within the age bracket of 30–50 years with

early menopause before the age of 50 years. This is in

marked contrast with the studies conducted in the West [26].

In the West, many risk factors have been well documented

for breast cancer, such as early menarche, advanced age at

Table 2 Observed SNPs in the coding and non-coding regions of the

SYK gene among Pakistani population with breast cancer

Nt. Alteration Exon/intron Status Percentage (%)

26,367 G[A Exon 4 Novel 68.3

26,414 G[A Exon 4 Novel 30.0

26,423 G[A Exon 4 Novel 56.6

26,353 G[A Intron 4 Novel 35.8

26,484 G[A Intron 5 Novel 49.1

26,531 G[A Intron 5 Novel 33.3

63,540 T[G Intron 9 Novel 80.7

Fig. 2 SSCP and DNA

sequencing results of exon 4 of

SYK gene. a SSCP analysis

showing band shift variation in

all disease samples. b PCR

product after sequencing

showed that SNP at splice

variant site at position 63,540

T[G, resulted from a G ? T

transversion. c Nucleotide

variation at position 63,540

T[G

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first full term pregnancy, parity, smoking, lactation and late

menopause, etc. [27], but such factors did not show any

correlation with breast cancer development in our society.

Apart from environmental and reproductive factors,

genetic instability also plays important role in breast cancer

development and metastasis [28]. The tumor suppressive

role of protein tyrosine kinase family has been well

reported in breast cancer tumorigenesis and metastasis

[15]. Syk tyrosine kinase is a key player and vital for its

role in breast cancer prognosis [29]. SYK is expressed in

normal epithelial cells of breast and also in relatively

benign breast cancer cells, but is missing from highly

metastatic cells due to methylation [14, 17]. In the present

study, seven novel mutations were identified in SYK gene

involved in breast cancer. Three were identified in three in

non-coding region of intron 4 and 5, one in splice donor

site of intron 9 and three in exon 4. In accordance with

earlier work, these identified SNPs did not show any sig-

nificant correlation with respect to early menarche, late

menopause, and older age [30]. Identified intronic SNPs

might have important role in regulating SYK gene

expression through intron-mediated enhancement phe-

nomena. But to determine the role of these mutations

within the intron will require further investigations.

A mutation at the 50splice donor site of intron 9 was

detected in 96/120 patients (80.8 %) with breast cancer and

was significantly associated with early menarche before the

age of 11 years (P \ 0.05). The nucleotide transformation

of T ? G at splice donor site, resulted in a GG formation.

This nucleotide alteration results in disruption of the cis-

acting splice regulatory site. Such changes result in failure

of snRNPs binding with splice site that leads to introns

retention [31, 32]. Retained introns in transcripts might

generate mutated Syk protein with consequences for breast

cancer development. Examination of association between

SYK splice variants and breast cancer will be very fruitful

for identification of new generation of biomarkers.

Table 3 Thermodynamic parameters of 50UTR secondary structures

predicted by UNAFOLD

Structure DG

(kcal mol-1)

DH

(kcal mol-1)

DS

(cal k-1 mol-1)

Tm �C

Original

SYK

-1.08 -37.7 113.32 59.5

Mutated

SYK

-0.03 -50.9 -157.42 50.2

26,367

G[A

-1.08 -37.7 113.32 59.5

26,414

G[A

-0.12 -36.8 -113.51 51.1

26,423

G[A

-1.08 -37.7 113.32 59.5

Fig. 3 In silico prediction of the 50UTR secondary RNA structure of

polymorphic SYK gene using UNAFOLD. a Secondary structure

of original 50UTR region of SYK mRNA. b Secondary structure of

mutated 50UTR region of SYK mRNA containing all of three

mutations. c Secondary structure with mutation 26,414 G[A. All of

these mutations were indentified in 43 mutants collectively

Mol Biol Rep (2012) 39:8345–8351 8349

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Three novel SNPs were identified in exon 4 encoding

the 50UTR of SYK gene (ENST00000375746). Among

these nucleotide alterations, the SNP at position 26,414

G[A was significantly associated with age at first full term

pregnancy (P \ 0.05). Unlike DNA, RNA-mediated

information is present in its secondary structure that gen-

erally recognizes RNA binding proteins [33]. The 50UTR

region generally contains genetic information for post-

transcriptional regulation of gene expression in term of

mRNA average half life, sub-cellular localization and its

translation efficiency [34]. Based on these observations, we

propose that any mutation within the 50UTR might affect

structural stability of SYK pre-mRNA - a major determi-

nant of gene regulation and expression [33, 35, 36].

In considering the importance of 50UTR region, bioin-

formatics analysis was carried out to analyze the effects of

the identified SNPs in the 50UTR region. Using the

UNAFOLD server, the structure of 50UTR region was

predicted with user defined parameters. Through structural

analysis, it was observed that the mutated structure con-

taining all three mutations collectively showed a larger

stem and loop instead of smaller ones with a longer tail.

The stem and loop of the mutated structure containing a

higher GC content (high percentage of triple hydrogen

bonding) as compared to the original structure. Through

physiochemical analysis, the mutated model showed high

values of DG and low Tm (-0.03 kcal mol-1 and 50.2 �C,

respectively compared to the wild type model with

-1.08 kcal mol-1 and 59.5 �C values (Table 3). These

structural variations and higher value of DG revealed

the mutated structure to be more stable compared to the

original one. As the stability of the 50UTR region increases,

more energy will be required to linearize it and interaction

of promoter binding protein might also be affected.

Furthermore, the relative distance of *60–70bps from

transcription start site (ATG) is also very crucial for

binding of transcription factors [37]. The relative distance

of our identified SNPs from start codon is 124, 75 and 68

bps, respectively. Thus, it may be speculated that the

identified SNP(s) may be responsible for down regulation

of SYK, resulting in uncontrollable cellular growth and

proliferation, eventually promoting breast cancer progres-

sion [38].

To analyze mutational effects individually, all three

identified SNPs in UTR region were also introduced indi-

vidually to assess their effect on UTR secondary structures.

Mutations at 26,367 G[A and 26,423 G[A did not show

any effect on structural stability of 50UTR (Table 2), but

was stabilized with the presence of nucleotide variation at

26,414 G[A (Table 2; Fig. 3c). This suggests that

sequence variation at 26,414 G[A may be responsible for

the structural stabilization observed in the triple mutant.

These predictions will need to be verified experimentally.

The novel findings of SYK gene polymorphisms in human

breast cancer patients and their potential effect on its own

down regulation need further exploration experimentally.

Clarification of the role of SYK gene polymorphisms in

breast cancer progression and metastasis will require a large

set of patient samples and could result in the development of

biomarkers for breast cancer prognosis and therapy.

Acknowledgments We are grateful to the patients who took part in

this study and to the NORI Hospital for providing blood samples. The

authors also appreciate the help from Dr. Faraz A Malik for detection

of germline mutations in SYK gene, Dr. Ramla Shahid for helpful

discussions in UTR structure analysis, Miss Hina Iqbal for her skillful

assistance and Miss Ruqia Mehmood Baig for her constant guidance

during the research period.

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